We have demonstrated that AAV-2 vectors can transduce human hepatocytes in vivo. However, AAV-mediated gene transfer is limited due to pre-existing humoral immunity and transgene expression is short-lived because of an immune response directed against the capsid containing hepatocytes. The goal of this project is to develop a novel class of human gene transfer vector based on adeno-associated virus (AAV) that combines high efficiency in vivo transduction of target tissues, with the capability to circumvent or overcome host immunologic responses in humans including the existence of neutralizing antibodies in the human population that may limit their efficacy. These novel vectors will be derived by 'molecular evolution' of DNA from an array of naturally occurring AAV serotypes. This array will comprise previously reported AAV variants from humans and primates, as well as new AAVs of non-primate origin whose isolation is our primary specific aim. The genomes of the various AAV isolates will be 'shuffled' to generate a DNA pool from which we will then generate a library of recombinant AAV particles having unique capsids. It is our idea that those capsids will combine the low reactivity with human sera as it is typical for non-primate AAVs, with the high efficiency of in vivo transduction as reported for primate/human AAVs. This AAV library will then be mixed with antibodies raised against primate/human AAV serotypes and used to transduce mouse liver in vivo. It is hoped that this form of selective pressure will enable us to isolate AAV capsid sequences allowing efficient in vivo transduction while evading neutralization from pre-existing immunity. We plan to test these new vectors in animal models of hemophilia. Our ultimate goal is to utilize any sequences that we can identify for development of novel improved AAV vectors for use in clinical trials.